Lab.

4 Differential stain

Objectives
.List types of differential stain *
Describe Gram stain, its method and *
.benefit of using it

Microbiology -‫ ا لتمريضا لثاني‬-‫عمليا لفصلا الول‬

Department of Microbiology Al-Hadi University College
Dr. Abd Al Munim Naji
 :Differential stain
It is a staining technique consists of more than
one dye used successively to identify
.organisms according to their type of reaction
 Types of differential stain
.Gram stain  for most type of bacteria and yeast
.Zeihl Neelsen (acid-fast) stain  for T.B
.Spore stain
.Capsule stain
 GRAM STAINING
.One of the most important staining method
.Widely used in bacteriology
Discovered in 1884 by Gram (a Danish physician)
Using two dyes in sequence. Each of different color, he found
:bacteria fall into two different categories
 
• A) Those that retained the first dye (crystal violet)
throughout the staining procedure, are known as
"Gram Positive".
• B) Those that lost the first dye (crystal violet ) after
washing with a decolorizing solution and stained
with the second dye (safranin or neutral red ) are
known as "Gram Negative".
GRAM POSITIVE
appear VIOLET

G+ve
G-ve

GRAM NEGATIVE
are RED in color.
The Benefit of Gram Staining
Technique:
1-Differentiate between bacteria of the same
morphology.

2-To determine the relative number, morphology

and staining reaction of bacteria in a smear taken
directly from a patient.
The Theory Behind this Differences in Bacterial
Reaction:
The differences in Gram Stain Reactions are due to the
differences in the biochemical composition of bacterial cell
walls.

Gram Positive cell walls: Teichoic, Teichuronic acid with

thick layer of peptidoglycan (protein-sugar complex)

Gram Negative cell walls: Thin layer of peptidoglycan with

multi layers of lipids.
Compeer between Gram positive and gram negative bacteria
Gram Staining Method:
1)Flood the slide with crystal violet.
violet Leave to act for 1-2 min wash w
tap water.

2) Apply Grams’ iodine (a mordant).

mordant Leave to act for one Minute.
Wash with tap water.

3) Apply 95% ethyl alcohol (a decolorizer).

decolorizer Leave to act for10-20
seconds. Wash with tap water.

4) Apply (safranin or neutral red) (the counter stain).

stain Leave to act fo
5 min., wash with tap water. Blot, dry in air and examine with oil
immersion lens.
Summary:
* Gram stain used to differentiate between bacteria of same
morphology .

* Gram positive bacteria appear violet in color and gram

negative bacteria appear red in color.
Zeihl Neelsen (acid-fast) stain  for T.B.

ZN stain AFB
Special Staining Ziehl Neelsen stain (acid fast stain)
• Procedure for Acid Fast Stain;
1) the specimen is taken from either a suspected patient with
tuberculosis, or from formalin killed culture, or from a non pathogenic
species of Mycobacterium, such as M. phlei. Make a heat fixed smear
from the specimen or the bacteria you obtain ( as shown earlier).

2) Flood with strong carbol fuchsin stain,

stain and steam for 5 minutes
3) Leave to cool down, hold the slide with a forceps in an angle position
and add 20% H2SO4 or acid alcohol until no more color runs off.

4) Wash with water and add the counter stain which is methylene blue.
blue
5) Wash with water, leave to dry in an angle position, examine by oil
immersion lens for red bacilli.
Mycobacterium tuberculosis

Acid – Fast (Kinyoun) Stain

Mycobacterium tuberculosis

ZN stain AFB
ziehl-neelsen.wmv